Bioengineered enzymes for the development of improved reagentless biosensors and their applications in food analysis

Dane projektu

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CSC - Cost-sharing contracts

Cel

Foreseen Results

Production and study of new 02-independent enzymes for the biosensor development is of high significance for analytical chemistry in both fundamental and applied aspects. First, it is related to the introduction of new PQQ-dependent dehydrogenases and other enzymes (with bound cofactors) which are obviously more suitable for biosensors than oxygen dependent ones. However, the commercial lack in these enzymes restricts their use in fundamental and applied studies. Second, characterization of new and genetically engineered PQQ-dependent dehydrogenases or other enzymes in this project can create the basis for their fundamental and practical studies in various biosensor configurations. Bioengineering of both non-02-dependent enzymes with bound cofactors addresses a very fundamental and significant issues as it contributes to several key issues : (i) how the structure of genes is transformed to the structure of enzymes and subsequently to their properties and (ii) how to practically control and produce enzymes with desirable structures and properties. Solution of these fundamental problems would strongly stimulate practical applications of biosensors through the production of more stable and even more specific enzymes. Electrochemical studies of new peroxidases are also a key for the development of a more specific detection of analytes using rather coupled peroxidase-oxidase reactions than only oxidase-direct registration of oxygen or peroxide. Additionally, an enhanced direct electron transfer between peroxidase and the electrode material would lead to the construction of highly sensitive mediatorless electrodes, which is of great practical importance. Moreover, the knowledge obtained on the bioelectrochemistry of peroxidases should be of great importance for further studies of other heme-containing enzymes in general having an even more complex structure then peroxidases.During the past decade more and more attention is paid to the design of enzymes possessing higher stability, reactivity, selectivity for their industrial application. Development of biosensors also badly need more stable and in some cases especially designed enzymes, e.g., for efficient electron transfer communication with electrodes. Bioengineering of enzymes for biosensor development is thus a possible way to solve some of the problems restricting practical application of biosensor. For these reasons the purpose of this proposal is directed to establish production of 02-independent redox enzymes enzymes (e.g., glucose dehydrogenase) and search for new type of redox enzymes (e .g., peroxydases) with increased rates of electron communication with electrodes.

The purpose of this project is :

1. to search for new microorganisms producing 02-independent oxidoreductases and new, more stable peroxidases,2. to produce and isolate 02-independent oxidoreductases and new types of peroxidases, including their characterization in homogeneous solutions,3. to produce genetically engineered 02-independent oxidoreductases using random and peroxidases using random and site specific mutagenesis,4. to synthesize new redox mediators for preparation of enzyme electrodes based on redox hydrogels and conducting polymers,5. to characterize electron transfer properties of natural and genetically engineered enzymes and preparation of amperometric electrodes,6. to integrate the electrodes into flow injection systems and to apply them for food quality control in dairy and beverage plants in Lithuania as well as for milk and beverage products from ordinary food stores in Lithuania and Moscow.